Application of Continuous Culture Methods to Recombinant Protein Production in Microorganisms

Peebo, Karl; Neubauer, Peter

doi:10.3390/microorganisms6030056

Cited by 53 publications

(60 citation statements)

References 92 publications

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“…In chemostat cultivations, the cells are maintained in a steady‐state growth environment by the supply of a constant medium flow with one or more final cell‐density‐limiting nutrients, and simultaneous removal of cultivation broth at a defined growth rate. Though, the commercial use of chemostat processes are limited, high yields of continuous recombinant protein production have been reported in the scientific literature (Peebo & Neubauer, 2018). Chemostat cultivations are also widely applied as a tool for functional genomics research and characterization of microbial physiology in a controlled and reproducible manner (Hoskisson & Hobbs, 2005; Jansen et al, 2005; Peebo & Neubauer, 2018).…”

Section: Introductionmentioning

confidence: 99%

“…Though, the commercial use of chemostat processes are limited, high yields of continuous recombinant protein production have been reported in the scientific literature (Peebo & Neubauer, 2018). Chemostat cultivations are also widely applied as a tool for functional genomics research and characterization of microbial physiology in a controlled and reproducible manner (Hoskisson & Hobbs, 2005; Jansen et al, 2005; Peebo & Neubauer, 2018). However, steady‐state chemostats exert a selective pressure on the cells, which may result in genetic or nongenetic adaptation or transition in the cellular state over time (Jansen et al, 2005; Mashego, Jansen, Vinke, van Gulik, & Heijnen, 2005; Seresht et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Fluctuations in glucose availability prevent global proteome changes and physiological transition during prolonged chemostat cultivations of Saccharomyces cerevisiae

Wright

Wulff

Palmqvist

et al. 2020

Biotech & Bioengineering

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Chemostat cultivation mode imposes selective pressure on the cells, which may result in slow adaptation in the physiological state over time. We applied a two‐compartment scale‐down chemostat system imposing feast–famine conditions to characterize the long‐term (100 s of hours) response of Saccharomyces cerevisiae to fluctuating glucose availability. A wild‐type strain and a recombinant strain, expressing an insulin precursor, were cultured in the scale‐down system, and analyzed at the physiological and proteomic level. Phenotypes of both strains were compared with those observed in a well‐mixed chemostat. Our results show that S. cerevisiae subjected to long‐term chemostat conditions undergoes a global reproducible shift in its cellular state and that this transition occurs faster and is larger in magnitude for the recombinant strain including a significant decrease in the expression of the insulin product. We find that the transition can be completely avoided in the presence of fluctuations in glucose availability as the strains subjected to feast–famine conditions under otherwise constant culture conditions exhibited constant levels of the measured proteome for over 250 hr. We hypothesize possible mechanisms responsible for the observed phenotypes and suggest experiments that could be used to test these mechanisms.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Fluctuations in glucose availability prevent global proteome changes and physiological transition during prolonged chemostat cultivations of Saccharomyces cerevisiae

Wright

Wulff

Palmqvist

et al. 2020

Biotech & Bioengineering

View full text Add to dashboard Cite

show abstract

“…Compared with the batch fermentation, continuous process is conducted by adding the substrate and harvesting the final product continuously and simultaneously, leading to economic advantages including the following: i) not requiring sterilization of facilities in each round, and the residual substrates can be recycled, cutting the energy and substrate costs; ii) prolonging the microbial exponential growth, reducing the processing time; and iii) eliminating the inhibition of toxic by‐products in a timely manner, ensuring high yields of the final product . The continuous fermentation processes have been applied successfully for production of ethanol, PHA, hydrogen, LA, and protein . It will be further developed for more products in NGIB.…”

Section: Next‐generation Industrial Biotechnologymentioning

confidence: 99%

“…[59,60,86] The continuous fermentation processes have been applied successfully for production of ethanol, [87] PHA, [64] hydrogen, [88] LA, [89] and protein. [90] It will be further developed for more products in NGIB.…”

Section: Ngib Based On Open Continuous and Intelligent Processesmentioning

confidence: 99%

Next‐Generation Industrial Biotechnology‐Transforming the Current Industrial Biotechnology into Competitive Processes

Chen

2019

Biotechnology Journal

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The chemical industry has made a contribution to modern society by providing cost‐competitive products for our daily use. However, it now faces a serious challenge regarding environmental pollutions and greenhouse gas emission. With the rapid development of molecular biology, biochemistry, and synthetic biology, industrial biotechnology has evolved to become more efficient for production of chemicals and materials. However, in contrast to chemical industries, current industrial biotechnology (CIB) is still not competitive for production of chemicals, materials, and biofuels due to their low efficiency and complicated sterilization processes as well as high‐energy consumption. It must be further developed into “next‐generation industrial biotechnology” (NGIB), which is low‐cost mixed substrates based on less freshwater consumption, energy‐saving, and long‐lasting open continuous intelligent processing, overcoming the shortcomings of CIB and transforming the CIB into competitive processes. Contamination‐resistant microorganism as chassis is the key to a successful NGIB, which requires resistance to microbial or phage contaminations, and available tools and methods for metabolic or synthetic biology engineering. This review proposes a list of contamination‐resistant bacteria and takes Halomonas spp. as an example for the production of a variety of products, including polyhydroxyalkanoates under open‐ and continuous‐processing conditions proposed for NGIB.

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“…Continuous culture systems allow for the long-term, controlled growth of microorganisms or cultured cells. Consequently, they have been used for many scientific and industrial uses, including producing biologics like small molecules [1–3] and recombinant proteins [4,5]; assessing the growth rate [6–9] or metabolism [10,11] of microorganisms or cultured cells under defined conditions; and for studying evolution [12–14]. Continuous culture systems typically operate in one of two modes: a chemostat, where a limited amount of nutrients are constantly added to the culture, or a turbidostat, where the culture density is kept constant [15].…”

Section: Introductionmentioning

confidence: 99%

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test

Ong

Pence

Molik

et al. 2020

Preprint

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Continuous culture systems allow for the controlled growth of microorganisms over a long period of time. Here, we develop a novel test for mutagenicity that involves growing yeast in continuous culture systems exposed to low levels of mutagen for a period of weeks. In contrast, most microorganism-based tests for mutagenicity expose the potential mutagen to the biological reporter at a high concentration of mutagen for a short period of time. Our test improves upon the sensitivity of the well-established Ames test by at least 20-fold for each of two mutagens that act by different mechanisms (the intercalator ethidium bromide and alkylating agent methylmethane sulfonate). To conduct the tests, cultures were grown in small, inexpensive continuous culture systems in media containing (potential) mutagen, and the resulting mutagenicity of the added compound was assessed via two methods: a canavanine-based plate assay and whole genome sequencing. In the canavanine-based plate assay, we were able to detect a clear relationship between the amount of mutagen and the number of canavanine-resistant mutant colonies over a period of one to three weeks of exposure. Whole genome sequencing of yeast grown in continuous culture systems exposed to MMS demonstrated that quantification of mutations is possible by identifying the number of unique variants across each strain but with lower sensitivity than the plate-based assay. In conclusion, we propose yeast grown in continuous culture systems can provide an improved and more sensitive test for mutagenicity.

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Application of Continuous Culture Methods to Recombinant Protein Production in Microorganisms

Cited by 53 publications

References 92 publications

Fluctuations in glucose availability prevent global proteome changes and physiological transition during prolonged chemostat cultivations of Saccharomyces cerevisiae

Fluctuations in glucose availability prevent global proteome changes and physiological transition during prolonged chemostat cultivations of Saccharomyces cerevisiae

Next‐Generation Industrial Biotechnology‐Transforming the Current Industrial Biotechnology into Competitive Processes

Yeast grown in continuous culture systems can detect mutagens with improved sensitivity relative to the Ames test

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